Wnt (wingless and int homologue) is a family of cysteine-rich secreted glycoproteins and has been identified in many vertebrates and invertebrates. They have been shown to have important roles for the decision of cell fate and behavior at multiple stages during development and cancer. They bind a family of specific receptors on the cell surface (Frizzled (Fz) family) and activate the cell signaling pathways to elicit their effects on, for example, gene transcription (Cadigan K M and Nusse R, 1997; Polakis P, 2000). One of the hallmarks of the Wnt family is the existence of 23 or 24 cysteine residues at conserved positions in the protein molecules. It has been assumed that these cysteine residues may have a critical role for folding of Wnt proteins by disulfide bond formation.
The processing and secretion of Wnt proteins was studied with cultured cells engineered to express various Wnts (Smolich B D, et al, 1993; Burrus L W and McMahon A P, 1995). The processing of Wnt is not efficient in most cell types, because multiple processing intermediates are present. Wnt is therefore not well secreted outside of the cells. Most of Wnt protein associates with a HSP70 protein, BiP, and is retained in the ER (Kitajewski J, et al, 1992). Screening for genes involved in Wg (Drosophila homologue) signaling by Drosophila deficiency kits has also identified a new gene(s), whose product(s) is required for the processing or secretion of Wg (Muller H, et al, 1999). These results indicate that the processing and secretion of Wnt are complex and that a number of specific factors are involved in these events.
One of the Drosophila segment polarity genes, porcupine (porc) encodes a multipass transmembrane ER protein, which is required for the normal distribution of Wg in embryos (Kadowaki T, et al, 1996). In porc mutant embryos, Wg is sequestered in its synthesizing cells and not distributed among the surrounding cells. Wg signaling components are well conserved in multicellular organisms, and porc homologs are also present in other species. C. elegans porc homolog mom-1 is necessary in Mom-2 producing cells as Porc is required in Wg-synthesizing cells (Thorpe C J, et al, 1997). Vertebrate (mouse and Xenopus) homologues of porc have been shown to modify the N-glycosylation of Wg and mouse Wnt proteins in cultured cells (Tanaka K, et al, 2000).
Recently, the human homologue of the Drosophila gene Porcupine (Porc) has also been cloned (Caricasole A, et al, 2002). The human Porcupine locus (PPN/MG61) spans 15 exons over approximately 12 kb of genomic sequence on Xp 11.23. Like its mouse and Xenopus homologues, PPN/MG61 is expressed in a tissue-specific fashion. Evidence also indicates that human PPN/MG61 can influence the activity of a human Wnt7A expression construct in a T-cell factor-responsive reporter assay. These results demonstrate that the porc gene family encodes the evolutionary conserved ER membrane proteins involved in the processing of the Wnt family.
Based on the amino acid sequence conservation between Porc and other membrane-bound acyltransferase superfamily, Porc is likely to function as the acyltransferase of Wnt (Hofmann K, 2000). Porc acylates Wnt proteins and anchors them at the ER membrane to stimulate their post-translational N-glycosylation, which is necessary for secretion. In the absence of porc, Wnt proteins are not secreted from the synthesizing cells, and therefore Wnt signaling is not activated in the surrounding cells.
All biochemically characterized members of the membrane-bound acyltransferase superfamily encode enzymes that transfer organic acids, typically fatty acids, onto hydroxyl groups of membrane-embedded targets. Examples include ACAT (cholesterol acyltransferase; transferring fatty acids to cholesterol), Are ½ (transferring fatty acids to yeast sterols), DGAT (diacylglycerol O-acyltranserase; transferring fatty acids to diacylglycerol), wax synthase (transferring fatty acids to long-chain alcohols), DltB (involved in the incorporation of alanine into lipoteichoic acids) and AlgI (involved in alginate O-acetylation) (Hofmann K, 2000). The presumed enzymatic role of the protein family is corroborated by the conservation of polar residues within and adjacent to the membrane. Most notably, a histidine positioned within a long hydrophobic region is invariant, making it a likely active-site residue.
Other members of the superfamily have been characterized only genetically. These include the Drosophila gene porcupine along with its nematode homolog mom-1 Porcupine is essential for Wingless signaling and affects Wingless processing and secretion (Kadowaki T, et al, 1996). Mom-1 was identified in a genetic screen for endoderm differentiation mutants, along with mom-2 (a wingless homolog) and mom-5 (a frizzled homolog) (Thorpe C J, et al, 1997). A role for the Porcupine-like proteins in Wingless signaling is well established. Based on the homology their mode of action as an acyltransferases appears plausible. However, the nature of the substrate is still an open question. These observations are not the only links between acylation and Wnt signaling. Members of the Dickkopf (DKK) family of Wnt inhibitors were found to be related to collapses (Aravind L and Koonin E V, 1998). Therefore, there is a hint that deacylation mediated by Dickkopf (DKK) proteins might be antagonistic to acylation mediated by Porcupine (Porc; PPN/MG61).
The Wnt/Frizzled receptor pathway involves important regulatory genes that carry polymorphisms associated with primary carcinomas. In the course of downstream signaling cytosolic β-catenin accumulates, translocates into the nucleus, and then enhances gene expression by complexing with other transcription factors. In the absence of Wnt signals, free cytosolic β-catenin is incorporated into a complex consisting of Axin, the adenomatous polyposis coli (APC) gene product, and glycogen synthase kinase (GSK)-3β. Conjunctional phosphorylation of Axin, APC, and β-catenin by GSK-3β designates β-catenin for the ubiquitin pathway and degradation by proteasomes.
It is known that Wnt/β-catenin signaling promotes cell survival in various cell types. Wnt signaling pathway is also thought to be associated with tumor development and/or progression. Aberrant activation of the Wnt signaling pathway is associated with a variety of human cancers. For example, mutations in the gene APC are an initiating event for both sporadic and hereditary colorectal tumorigenesis. APC mutants are relevant in tumorigenesis, since the aberrant protein is an integral part of the Wnt-signaling cascade. The protein product contains several functional domains acting as binding and degradation sites for β-catenin. Mutations that occur in the amino-terminal segment of β-catenin are usually involved in phosphorylation-dependent, ubiquitin-mediated degradation and, thus, stabilize β-catenin. When stabilized cytoplasmic-catenin accumulates, it translocates to the nucleus interacting with the Tcf/Lef high-mobility group of transcription factors that modulate expression of oncogenes such as c-Myc. Evidence has also shown that Wnt proteins are over-expressed and aberrantly activated in a variety of cancers. However, the role of the Wnt signaling pathway in oncogenesis remains unclear.
The method of the present invention demonstrates that human homologue of Drosophila Porcupine gene, PPN/MG61, is expressed abundantly in a variety of human cancer cell lines, including lung cancer, breast cancer, colorectal cancer, mesothelioma, head and neck cancer and melanoma. In contrast, PPN/MG61 expression was not observed in cultured normal cells. The present method further demonstrates that the PPN/MG61 are over-expressed in primary lung cancer and mesothelioma tissue samples, which was not expressed in their matched normal tissue samples (from same patients). In addition, PPN/MG61 expression was detected in serum from patients of a variety of cancers, including lung cancer and thyroid cancer, etc, but not in normal serum samples.
The method of the present invention also demonstrates the overexpression of human PPN/MG61 is necessary for survival of cancer cells. Knocking-down PPN/MG61 mRNA and/or inhibiting the acyltransferase activity of PPN/MG61 induce apoptosis in many types of cancers, especially lung cancer and mesothelioma. These data suggest that lipid modification of the Wnt signaling molecules by PPN/MG61 is important for function of the pathway in tumorigenesis, and that PPN/MG61 could be a provocative therapeutic target. Thus, detection of PPN/MG61 expression and inhibition of PPN/MG61 enzyme activity may have widespread implications for molecular diagnosis, early detection and treatment of human cancers.